Mammary epithelial cells undergo ductal morphogenesis only in compliant 3D extracellular matrices (floating collagen gels or Matrigel), but not in otherwise identical matrices that are stiff (attached 3D collagen gels) or have increased collagen density. An understanding of how cancer cells interact with local tissue environments requires the ability to observe the relationship between signaling molecules, subcellular structures, and components of the ECM within a 3D environment. Few studies have investigated signaling events in 3D, and to our knowledge none have done so using live cell imaging or FRET approaches. 3D imaging adds complexity to these fluorescence studies, including the inherent challenge of a 3D volume as opposed to a 2D image, problems of overlapping spectra, low signals, and light scatter. The goal of this project is to develop imaging techniques in the context of 3D culture systems to directly visualize signaling pathways relevant to breast tumor cell behavior. Our underlying hypothesis is that the Rho signaling pathway is the sensing mechanism by which the physical properties of the microenvironment are conveyed to signaling effectors within the cytoskeleton. We propose the following Aims: Aim 1: Characterize endogenous signals of the 3D matrix and tissue environment within which we wish to investigate signaling events A) Characterize the multidimensional signals of endogenous fluorophores within cells within a 3D collagen matrix of various densities and compositions The "fingerprint" of endogenous fluorophores (NADH, FAD, and collagen) within collagen matrices will be mapped spatially using multiphoton laser-scanning microscopy (MPLSM) and Spectral and Fluorescent Lifetime Microscopy (SLIM and FLIM). B) Characterize the endogenous signals in the mouse mammary gland during tumorigenesis Endogenous signals (NADH, FAD, and collagen) will be characterized by MPLSM, SLIM, and FLIM in models of mouse mammary tumorigenesis, generating novel data regarding metabolic changes during tumorigenesis. Aim 2: Develop strategies for directly visualizing Rho/ROCK signaling pathways within 3D matrices using Multiphoton Laser-Scanning microscopy (MPLSM) and Fluorescent Lifetime Microscopy (FLIM) A) Investigate the matrix-dependent activation of Rho GTPase. Fluorescent probes coupled to Rho and a Rho binding domain will be used as FRET pairs to determine Rho activation spatially and temporally in a 3D context within collagen gels and definable microfluidic channels. B) Apply FRET/FLIM techniques to characterize the coupling of Rho to its effector ROCK in response to collagen density. Coupling of activated Rho to ROCK, and regulation of myosin-mediated contractility will be determined spatially and temporally within 3D collagen matrices.